Immuno-positron emission tomography (PET) is an imaging tool useful in diagnostic, prognostic and therapeutic oncology. Monoclonal antibodies (mAbs) uniquely bind specific antigens produced by tumors, allowing researchers to establish the presence, location, and number of neoplastic foci. MAbs can be produced in nearly unlimited quantities with absolute specificity using the hybridoma method. Treatment using mAbs can have different forms: the mAbs can be administered alone or can be combined with a radioactive molecule, which then provides radiation directly to the tumor cells. The labeling of antibodies with PET isotopes provides not just better sensitivity but also superior resolution. In immuno-PET, mAbs are labeled with various PET radionuclides and used in cancer patients who produce tumor-associated antigens.
Various radionuclides have been used for PET but most of them have limitations that make them unattractive for PET. Direct labeling methodologies using 76Br (t1/2=17 hours) and 124I (t1/2=4 days) encounter the difficulties like loss of immunoreactivity, random radiolabel incorporation and dehalogenation. Indirect labeling using metal such as 64Cu (t1/2 of 12.7 hours) are superior to the direct labeling methodologies but also have their shortcomings such as demetallation and a 36 hour maximal image time.
Zirconium-89 (89Zr; t1/2, =78.4 h, β+: 22.8%) is widely used in immuno-PET applications because its half-life pairs well with the biological half-life of a circulating monoclonal antibody (mAb), and because it can be routinely produced in high specific activity, high radiochemical purity and can be shipped around the world. It is produced by a 89Y(p,n) 89Zr reaction and its decay produces two 511 KeV photons that are used in PET meaning that there is no interfering γ-rays for PET detection.
In clinical immuno-PET applications the acyclic iron chelator desferrioxamine B (DFO) is used exclusively to attach 89Zr to a mAb. FIG. 15 shows 89Zr to a mAb via the chelator DFO.
Despite the exclusive use of this chelator, it is not optimal for 89Zr-immuno-PET since the 89Zr4+ prefers ligands that form eight coordinate complexes while DFO can only form a 6 coordinate complex leaving 2 coordination sites vacant (shown by the Xs in the above structure), making it vulnerable to transchelation by endogenous proteins once injected in vivo. The 89Zr DFO chelation product is in many instances unstable.
Moreover, 89Zr-transchelation is observed as elevated levels of radioactivity in the liver and kidney that eventually localize to the bones of animals receiving the radiotracer. This non-specific uptake complicates the biodistribution and clearance profile of new mAbs and retards the critical processes that identify the most promising mAbs to be carried forward into clinical trials. The need for 89Zr chelators with improved stability remains an important and active area of research.
The current strategy to prepare clinical 89Zr-radiopharmaceuticals relies on the reaction of 89Zr-oxalate (89Zr—OX) with the bifunctional chelator (BFC), desferrioxamine B (DFO), and its analogues, which are derivatives of the siderophore desferral, a growth promoting agent secreted by Streptomyces pilosus. Additionally, several second generation 89Zr chelators rely on the use of 89Zr—OX as a radiochemistry precursor. However, the use of an 89Zr—OX as a zirconium source has always been viewed as problematic with the current strategy producing 89Zr-mAbs with radiochemical yields, purities and specific activities that are not optimal. Recently, others have described efforts to optimize the production of 89Zr-mAbs, and although they were able to reduce the amount of mAb used in each radiochemical reaction while maintaining excellent radiochemical purity and specific activities that are comparable to that achieved using alternative strategies, the methodology still relied on the use of 89Zr—OX, which has many of the problems disclosed below.
Drawbacks to the present technologies used for 89Zr is that oxalate (or oxalic acid) is used in the purification of 89Zr, and the zirconium source currently for 89Zr-radiopharmaceutical preparation is 89Zr-oxalate. The barriers to using oxalate is that it is highly toxic, it decalcifies the blood, Ca-oxalate obstructs kidney tubules causing kidney stones, and it must be removed by purification before formulating the 89Zr-radiopharmaceutical for injection. This processing increases the time and handling exposure. Moreover, the reaction success using oxalate is dependent on the volume of the reaction. Thus, it is desired to not rely on oxalate for purification of 89Zr. Others have proposed metathetical conversion to the chloride ion from oxalate. However, older traditional methods of this metathetical conversion are cumbersome and require very harsh conditions like elevated temperatures, the use of concentrated acids and peroxide, specialized techniques like vacuum sublimation, difficulties in automating the process and finally, enhanced exposure to radiation. More recently some improvements have been made but the methods are still not optimal. The newer methods still require elevated temperatures for reagent drying, relatively long reaction/purification times, difficulties in automating the process and still enhanced radiation exposure.
Significant effort has been expended on 89Zr chelator development, and several new ligand classes have been reported. However, none of these new ligands produce 89Zr-complexes that are superior to 89Zr-DFO in vivo. Moreover, others have described a tris(hydroxypyridinone) ligand that was conjugated to Trastuzumab (TmAb) and radiolabeled with 89Zr. However, studies in vivo revealed significant instability when compared with 89Zr-DFO-trastuzumab and demonstrated three fold more radioactivity localized in the bones of mice receiving the hydroxypyridinone-based radiotracer. Accordingly, little progress has been made in identifying ligands that bind 89Zr to produce radiometal complexes that are superior to 89Zr-DFO, which currently is considered to be the “gold standard” in 89Zr-PET radiopharmaceutical applications.
Replacing 89Zr—OX with a different 89Zr precursor has never been evaluated in the preparation of 89Zr-immuno-PET agents.
It is with these problems and needs in mind that the present invention was developed.